We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University. The support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University and the Northwestern University Structural Biology Facilities is acknowledged. The Gatan K2 direct electron detector was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. We also thank the following facilities at Northwestern University: the Keck Interdisciplinary Surface Science Facility, the Structural Biology Facility, the Biological Imaging Facility, the Center for Advanced Molecular Imaging, and the Analytical Bionanotechnology Equipment Core. This research was supported by the National Science Foundation grant 1453576, the National Institutes of Health Director&#39;s New Innovator Award 1DP2HL132390-01, the Center for Regenerative Nanomedicine Catalyst Award and the 2014 McCormick Catalyst Award. SDA was supported in part by NIH predoctoral Biotechnology Training Grant T32GM008449.

1. Synthesis of Poly(ethylene glycol)-block-poly(propylene sulfide)-Thiol
<ol>
	<li>Synthesize methoxy-poly(ethylene glycol) mesylate (Mn: 750) (MeO-PEG17-Ms, I).
	<ol>
		<li>Dissolve 10 g of MeO-PEG17-OH in 200 mL of 100% toluene within a 3-neck round bottom flask (RBF) under magnetic stirring at 600 rpm.</li>
		<li>Connect the 3-neck RBF to a Dean-Stark apparatus, itself attached to a condenser, keep the entire system under inert gas, either nitrogen or argon.</li>
		<l...

<ol>
	<li>Stano, A., Scott, E. A., Dane, K. Y., Swartz, M. A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+T+cell+immunity+towards+a+protein+antigen+using+polymersomes+vs.+solid-core+nanoparticles.">Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles.</a> Biomaterials. 34 (17), 4339-4346 (2013).</li><li>Discher, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes:+tough+vesicles+made+from+diblock+copolymers.">Polymersomes: tough vesicles made from diblock copolymers.</a> Science. 284 (5417), 1143-1146 (1999).</li><li>Vasdekis, A. E., Scott, E. A., O'Neil, C. P., Psaltis, D., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+intracellular+delivery+based+on+optofluidic+polymersome+rupture.">Precision intracellular delivery based on optofluidic polymersome rupture.</a> ACS Nano. 6 (9), 7850-7857 (2012).</li><li>Yi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Nanostructure+Morphology+for+Enhanced+Targeting+of+Dendritic+Cells+in+Atherosclerosis.">Tailoring Nanostructure Morphology for Enhanced Targeting of Dendritic Cells in Atherosclerosis.</a> ACS Nano. 10 (12), 11290-11303 (2016).</li><li>Shum, H. C., Kim, J. W., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+fabrication+of+monodisperse+biocompatible+and+biodegradable+polymersomes+with+controlled+permeability.">Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability.</a> Journal of the American Chemical Society. 130 (29), 9543-9549 (2008).</li><li>Pessi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-assisted+engineering+of+polymeric+microcapsules+with+high+encapsulation+efficiency+for+protein+drug+delivery.">Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.</a> International Journal of Pharmaceutics. 472 (1-2), 82-87 (2014).</li><li>O'Neil, C. P., Suzuki, T., Demurtas, D., Finka, A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+encapsulation+of+biomolecules+into+polymersomes+via+direct+hydration.">A novel method for the encapsulation of biomolecules into polymersomes via direct hydration.</a> Langmuir. 25 (16), 9025-9029 (2009).</li><li>Saad, W. S., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+formation+by+flash+nanoprecipitation.">Principles of nanoparticle formation by flash nanoprecipitation.</a> Nano Today. 11 (2), 212-227 (2016).</li><li>Johnson, B. K., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+rapid+self-assembly+of+block+copolymer+nanoparticles.">Mechanism for rapid self-assembly of block copolymer nanoparticles.</a> Physical Review Letters. 91 (11), 118302 (2003).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+confined+impingement+jets+mixer+for+flash+nanoprecipitation.">A simple confined impingement jets mixer for flash nanoprecipitation.</a> Journal of Pharmaceutical Sciences. 101 (10), 4018-4023 (2012).</li><li>Allen, S., Osorio, O., Liu, Y. G., Scott, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+assembly+and+loading+of+theranostic+polymersomes+via+multi-impingement+flash+nanoprecipitation.">Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation.</a> Journal of Controlled Release. 262, 91-103 (2017).</li><li>Bobbala, S., Allen, S. D., Scott, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+nanoprecipitation+permits+versatile+assembly+and+loading+of+polymeric+bicontinuous+cubic+nanospheres.">Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres.</a> Nanoscale. 10 (11), 5078-5088 (2018).</li><li>Allen, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes+scalably+fabricated+via+flash+nanoprecipitation+are+non-toxic+in+non-human+primates+and+associate+with+leukocytes+in+the+spleen+and+kidney+following+intravenous+administration.">Polymersomes scalably fabricated via flash nanoprecipitation are non-toxic in non-human primates and associate with leukocytes in the spleen and kidney following intravenous administration.</a> Nano Research. , (2018).</li><li>Karabin, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+micellar+delivery+via+inducible+transitions+in+nanostructure+morphology.">Sustained micellar delivery via inducible transitions in nanostructure morphology.</a> Nature Communications. 9 (1), 624 (2018).</li><li>Mascoli, C. C., Weary, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limulus+amebocyte+lysate+(LAL)+test+for+detecting+pyrogens+in+parenteral+injectable+products+and+medical+devices:+advantages+to+manufacturers+and+regulatory+officials.">Limulus amebocyte lysate (LAL) test for detecting pyrogens in parenteral injectable products and medical devices: advantages to manufacturers and regulatory officials.</a> Journal of the Parenteral Drug Association. 33 (2), 81-95 (1979).</li><li>Pustulka, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+nanoprecipitation:+particle+structure+and+stability.">Flash nanoprecipitation: particle structure and stability.</a> Molecular Pharmaceutics. 10 (11), 4367-4377 (2013).</li><li>Tang, C., Amin, D., Messersmith, P. B., Anthony, J. E., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+directed+self-assembly+of+pH-responsive+antioxidant+nanoparticles.">Polymer directed self-assembly of pH-responsive antioxidant nanoparticles.</a> Langmuir. 31 (12), 3612-3620 (2015).</li><li>Gindy, M. E., Panagiotopoulos, A. Z., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+block+copolymer+stabilized+nanoparticles:+simultaneous+encapsulation+of+organic+actives+and+inorganic+nanostructures.">Composite block copolymer stabilized nanoparticles: simultaneous encapsulation of organic actives and inorganic nanostructures.</a> Langmuir. 24 (1), 83-90 (2008).</li></ol>

We have provided detailed instructions for the rapid fabrication of polymersomes using PEG17-bl-PPS35-SH as the diblock copolymer. Vesicular polymersomes are the primary aggregate morphology assembled at this ratio of hydrophilic PEG and hydrophobic PPS block molecular weight. When impinged multiple times, they have a diameter and polydispersity that matches polymersomes extruded through a 200 nm membrane after being formed via thin film hydration. This protocol thus eliminates the...

Nanocarriers allow for the controlled delivery of small and macromolecular cargo, including active entities that, if not encapsulated, would be either highly degradable and/or too hydrophobic for administration in vivo. Of the nanocarrier morphologies regularly fabricated, polymeric vesicles analogous to liposomes (also called polymersomes) offer the ability to simultaneously load hydrophilic and hydrophobic cargo1,2. Despite their promising advantages, polymersomes are still rare in clinical applications due, in part, to several key challenges in their manufacturing. For clinical use, polymersome formulations need to be made in large-scale, sterile, and consistent batches.
A number of techniques can be used to form polymersomes from a diblock copolymer, such as poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS), that include solvent dispersion3, thin film rehydration1,4, microfluidics 5,6, and direct hydration7. Solvent dispersion involves long incubation times in the presence of organic solvents, which may denature some bioactive payloads, like proteins. Thin film rehydration does not offer control over the polydispersity of the formed polymersomes, often requiring expensive and time-consuming extrusion techniques to achieve acceptable monodispersity. Furthermore, both microfluids and direct hydration are difficult to scale up for larger production volumes. Of the different nanocarrier fabrication methods, flash nanoprecipitation (FNP) offers the ability to make large-scale and reproducible formulations8,9,10. While FNP was previously reserved for the formulation of solid-core nanoparticles, our lab has recently expanded the use of FNP to include the consistent formation of diverse PEG-bl-PPS nanostructure morphologies11,12, including polymersomes11 and bicontinuous nanospheres12. We found that FNP was capable of forming monodisperse formulations of polymersomes without the need for extrusion, resulting in superior polydispersity index values compared to non-extruded polymersomes formed by thin film rehydration and solvent dispersion11. Bicontinuous nanospheres, with their large hydrophobic domains, were not able to be formed by thin film rehydration, despite forming under a number of solvent conditions with FNP12.
Here, we provide a detailed description for the synthesis of the PEG-bl-PPS diblock copolymer used in polymersome formation, the confined impingement jets (CIJ) mixer used for FNP, the FNP protocol itself, and the implementation of an automated system to reduce user variability. Included is information on how to sterilize the system sufficiently to produce endotoxin-free formulations for use in vivo, and representative data concerning the characterization of polymersomes formed by FNP. With this information, readers with interest in utilizing polymersomes for in vitro and in vivo work will be able to fabricate their own sterile, monodisperse formulations. Readers with experience in nanocarrier formulations and with polymer synthesis expertise will be able to rapidly test their own polymer systems using FNP as a potential alternative to their current formulation techniques. Additionally, the protocols described herein may be used as educational tools for the formulation of nanocarriers in nanotechnology laboratory courses.

<table>
	<tbody>
		<tr>
			<td>CanaKit Raspberry Pi 3 Ultimate Starter Kit - 32 GB Edition</td>
			<td>CanaKit</td>
			<td>UPC 682710991511</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Bearing Platform (Small) - 8mm Diameter</td>
			<td>Adafruit</td>
			<td>1179</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Motion 8 mm Shaft, 330 mm Length, Chrome Plated, Case Hardened, Metric</td>
			<td>VXB</td>
			<td>kit11868</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Rail Shaft Guide/Support - 8 mm Diameter</td>
			<td>Adafruit</td>
			<td>1182</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual-Position Precision Slide 4.5&#34; Stroke, 15 lb load capacity</td>
			<td>McMaster-Carr</td>
			<td>5236A16</td>
			<td></td>
		</tr>
		<tr>
			<td>MTPM-P10-1JK43 Iron Horse DC motor</td>
			<td>Iron Horse</td>
			<td>MTPM-P10-1JK43</td>
			<td></td>
		</tr>
		<tr>
			<td>Official Raspberry Pi Foundation 7&#34; Touchscreen LCD Display</td>
			<td>Raspberry Pi</td>
			<td>B0153R2A9I (ASIN)</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoBorg Reverse - Advanced motor control for Raspberry Pi</td>
			<td>PiBorg</td>
			<td>BURN-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pololu Carrier with Sharp GP2Y0D810Z0F Digital Distance Sensor 10cm</td>
			<td>Pololu</td>
			<td>1134</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruland PSR16-5-4-A Set Screw Beam Coupling, Polished Aluminum, Inch, 5/16&#34; Bore A Diameter, 1/4&#34; Bore B Diameter, 1&#34; OD, 1-1/4&#34; Length, 44 lb-in Nominal Torque</td>
			<td>Ruland</td>
			<td>PSR16-5-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol monomethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>202495</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanesulfonyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>471259</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene, Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite 545 (Diatomaceous Earth)</td>
			<td>Sigma Aldrich</td>
			<td>419931</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>320269</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma Aldrich</td>
			<td>791776</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T30805</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Sigma Aldrich</td>
			<td>360589</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum oxide, neutral, activated, Brockmann I</td>
			<td>Sigma Aldrich</td>
			<td>199974</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium methoxide solution, 0.5 M in methanol</td>
			<td>Sigma Aldrich</td>
			<td>403067</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene sulfide</td>
			<td>Sigma Aldrich</td>
			<td>P53209</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>320390</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free water</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30529.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper pH strips</td>
			<td>Fisher Scientific</td>
			<td>13-640-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free Dulbecco&#39;s PBS</td>
			<td>Sigma Aldrich</td>
			<td>TMS-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-4</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL all-plastic syringe</td>
			<td>Thermo Scientific</td>
			<td>S75101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sepharose CL-6B</td>
			<td>Sigma Aldrich</td>
			<td>CL6B200</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid chromatography column</td>
			<td>Sigma Aldrich</td>
			<td>C4169</td>
			<td></td>
		</tr>
		<tr>
			<td>CIJ mixer, HDPE</td>
			<td>Custom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-Blue hTLR4</td>
			<td>InvivoGen</td>
			<td>hkb-htlr4</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW-Blue Cells</td>
			<td>InvivoGen</td>
			<td>raw-sp</td>
			<td></td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td></td>
		</tr>
		<tr>
			<td>PYROGENT Gel Clot LAL Assays</td>
			<td>Lonza</td>
			<td>N183-125</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, scalable assembly and loading of bioactive proteins and immunostimulants into diverse synthetic nanocarriers via flash nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Here, we have presented a simple protocol for the formulation of nanocarriers capable of loading hydrophilic and hydrophobic cargo that are safe for in vivo mouse and non-human primate administration11,13. We have also included a detailed protocol for the synthesis of the polymer used in our representative results, along with a description for the fabrication of a custom instrument for the mechanically-controlled impingem...

Watch this Scientific Journal Video about Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation at JoVE.com

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

We believe that the use of flash nanoprecipitation to assemble complex nanostructures will allow researchers who do not have extensive experience in chemistry, engineering or nanomaterial science to fabricate advanced drug delivery systems independently for their own specific applications and needs. The main advantage of this technique is the generation of large volumes of diverse nanostructures at high concentration, low polydispersity, and loaded with bioactive molecules including small molecule drugs and proteins all formed under sterile conditions. Generally, individuals new to polymer synthesis may struggle because the polypropylene sulfide component is highly sensitive to oxidation by exposure to air.Visual demonstration of this method is critical as the multiple impingement steps are difficult to learn because they need to be performed very quickly in succession. To sterilize the confined impingement jets, or CIJ mixer, work within a biological safety cabinet to submerge the mixer with all parts disassembled into zero point one molar sodium hydroxide overnight. Reassemble the CIJ mixer and flow endotoxin-free water through the mixer using luerlock syringes, test the ph of the water, and continue to flow water through until the ph registers as neutral.To prepare impingement solution one, first weigh 20 milligrams of the synthesized polymer into a one point five milliliter tube. Then, add hydrophobic dyes, drugs, or other cargo. Finally, add 500 microliters of 100%tetrahydrofuran to the polymer and cargo, vortex vigorously to dissolve.To prepare impingement solution two, dissolve the hydrophilic cargo to be loaded within the polymer vesicles in 500 microliters of an aqueous buffer. Add 2.5 milliliters of an aqueous buffer of choice to a suitably sized reservoir. Now, place the reservoir under the CIJ mixer such that the outflow from the mixer directly enters the reservoir.Load the impingement solutions into separate one milliliter plastic disposable syringes, impinge the solutions against each other to simultaneously form nanostructures by inserting syringes into luerlock adapters at the top of the CIJ mixer. In a single, smooth, and rapid motion, depress both syringes simultaneously and with equal force. If multiple impingements are going to be performed, it is critical that they are done as quickly as possible to reduce the loss of valtower organic solvent.To perform multiple impingements, split the nascent nanostructure solution between two syringes and repeat this step up to four more times. Collect the outflow in the aqueous buffer filled reservoir and gently stir to ensure mixing. There are several options to remove the unloaded cargo and organic solvent.The nanocarrier formulation can be removed through excise exclusion or desalting column, through a tangential flow filtration system, through vacuum desiccation, or through dialysis as demonstrated here. Dialyze the nanocarrier formulation in the same aqueous buffer and reservoir used for impingement. Use tubing of an appropriate molecular weight cutoff for at least 24 hours, with at least two buffer changes.Once the unloaded cargo has been removed from the organic solvent, concentrate the nanocarrier formulation using a spin concentrator system following the manufacturer's instructions. Nanocarriers can be stored at four degrees celsius for weeks to months. Prior to use after storage, nanocarrier formulations should be briefly vortexed.Perform fabrication of a high-speed syringe pump as described in the text protocol. To use autorun mode, select autorun from the main menu. The system will prompt users to allow the motor to automatically position the syringe expulsion platform to the beginning of the precision slide.Ensure that the path in front of and behind the metal plate is clear prior to proceeding. Load the CIJ mixer into the rectangular opening of the rear expulsion carriage. Then, load one milliliter plastic syringes as before and mount syringes onto the female lure connectors of the CIJ mixer.Prior to running the instrument, set the desired motor speed and check that the system is clear as described in the text protocol. To expel reactants from the syringes and into the CIJ mixer, press the run button in the software interface. Representative cryogenic transmission electron micrographs demonstrate smaller and more mono-dispersed polymersomes after repeated impingements.Dynamic light scattering analysis confirmed a leftward shift of the size distribution of the polymersomes after repeated impingements. After five repeated impingements, the average diameter and polydispersity of the polymersomes match that of the extruded samples formed by thin film rehydration. Flash nanoprecipitation can be used to load a variety of hydrophobic and hydrophilic small molecules and macromolecules in polymersomes.Though this method can provide insight into how immunotherapies and vaccines function, it can also be applied to the study of how polymers self assemble into intricate nanostructures. Following this procedure, other methods like intravenous or subcutaneous administration to animal models can be performed in order to determine which cell subsets internalize the nanoparticles or what the route of clearance is for different nanostructures. The implications of this technique extend toward therapy of heart diseases and cancer as well as the development of vaccines for infectious diseases because these nanocarriers can target multiple drugs to very specific immune cells.

rapid-scalable-assembly-loading-bioactive-proteins-immunostimulants

Research

JoVE Journal

Bioengineering

7.8K visualizações.  Northwestern University. We believe that the use of flash nanoprecipitation to assemble complex nanostructures will allow researchers who do not have extensive experience in chemistry, engineering or nanomaterial science to fabricate advanced drug delivery systems independently for their own specific applications and needs. The main advantage of this technique is the generation of large volumes of diverse nanostructures at high concentration, low polydispersity, and loaded with bioactive molecules including small mol...